Antibiotic GE 2270 factors B1, B2, C1, C2, D1, D2, E and T

ABSTRACT

The present invention is directed to new antibiotic substances denominated antibiotic GE 2270 factors B 1 , B 2 , C 1 , C 2 , D 1 , D 2 , E and T, the addition salts thereof, the pharmaceutical compositions thereof and their use as medicaments, particularly in the treatment of infectious diseases involving microorganisms susceptible to them and their use as animal growth promoters.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation of application Ser. No. 08/311,288, filed Sep. 23, 1994 now abandoned, which is a continuation of application Ser. No. 08/144,102, filed Oct. 27, 1993, now abandoned, which is a continuation of application Ser. No. 08/000,920, filed Jan. 6, 1993, now abandoned, which is a continuation of application Ser. No. 07/856,857, filed Mar. 24, 1992, now abandoned, which is a continuation of application Ser. No. 07/665,612, filed Mar. 5, 1991, now abandoned, herein incorporated by reference.

The present invention is directed to new antibiotic substances denominated antibiotic GE 2270 factors B₁, B₂, C₁, C₂, D₁, D₂, E and T, the addition salts thereof, the pharmaceutical compositions thereof and their use as medicaments, particularly in the treatment of infectious diseases involving microorganisms susceptible to them.

The compounds of the invention are also active as growth promotant agents in animals, such as poultry, swine, ruminants, etc.

The compounds of the invention are isolated from the cultures of Planobispora rosea ATCC 53773 or an antibiotic GE 2270 producing variant or mutant thereof. In particular, they are found in the mycelium and also in the fermentation broths of the cultured microorganism.

Planobispora rosea ATCC 53773 was isolated from a soil sample which was deposited on Jun. 14, 1988 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852 Maryland, U.S.A., under the provisions of the Budapest Treaty.

The strain has been accorded accession number ATCC 53773.

This strain was already described in European Patent Application Publication No. 359062 (corresponding to U.S. patent application Ser. No. 401,278) in connection with the preparation of a new antibiotic substance named antibiotic GE 2270 factor A.

The compounds of the invention are produced by the above named microbial strains together with antibiotic GE 2270 factor A, which is normally the most abundant fraction.

The production of antibiotic GE 2270 factors B₁, B₂, C₁, C₂, D₁, D₂, E and T, is achieved by cultivating a Planobispora strain capable of producing it, i.e. Planobispora rosea ATCC 53773 or an antibiotic GE 2270 producing variant or mutant thereof, under aerobic conditions in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen, and inorganic salts. Many of the nutrient media usually employed in the fermentation art can be used, however certain media are preferred. Preferred carbon sources are glucose, mannose, galactose, starch, corn meal and the like. Preferred nitrogen sources are ammonia, nitrates, soybean meal, peptone, meat extract, yeast extract, tryptone, aminoacids, and the like. Among the inorganic salts which can be incorporated in the culture media there are the customary soluble salts capable of yielding sodium, potassium, iron, zinc, cobalt, magnesium, calcium, ammonium, chloride, carbonate sulfate, phosphate, nitrate and the like ions.

Ordinarily, the antibiotic-producing strain is pre-cultured in a shake flask, then the culture is used to inoculate jar fermentors for production of substantial quantities of the antibiotic substances. The medium used for the pre-culture can be the same as that employed for larger fermentations, but other media can also be employed. The antibiotic GE 2270 producing-strain can be grown at temperatures between 20° and 40° C., preferably between 24° and 35° C.

During fermentation, the antibiotic production can be monitored by testing broth or mycelial extract samples for antibiotic activity for instance by bioassays or TLC or HPLC procedures.

Sensitive organisms to antibiotic GE 2270 such as Bacillus subtilis and S. aureus can be used as test organisms. The bioassay is conveniently performed by the agar diffusion method on agar plates. Maximum production of antibiotic activity generally occurs between the second and the eighth day of fermentation.

Antibiotic GE 2270 factors B₁, B₂, C₁, C₂, D₁, D₂, E and T, are produced by cultivating the strain Planobispora rosea ATCC 53773, or an antibiotic GE 2270 producing mutant or variant thereof, and are mainly found in the mycelium even if a certain amount of product can be isolated from the fermentation broth.

Morphological Characteristics of Planobispora rosea ATCC 53773

Planobispora rosea ATCC 53773 grows well on most standard media. The vegetative mycelium forms long and irregularly branched filaments (0.5 to 1.0 micrometer) penetrating the agar and forming a compact growth on its surface. The mycelium remains unfragmented whether grown in liquid or in solid media. Its color ranges from light coral to pink coral on most of the tested media. The aerial mycelium is formed of long, wavy and slender hyphae with few lateral branches and grows in the air essentially parallel to the agar surface.

The aerial mycelium, when present, has a white-pink color. Sporangia are formed singly or in groups along the hyphae of the aerial mycelium and are about 6.0 to 8.0 micron long and 1.0 to 1.2 micron wide. They are attached to the hypha by a short sporangiophore (1.0 to 2.0 micrometer long). A longitudinal pair of fusiform straight spores (3.0 to 3.5×1.0 to 1.2 micrometer) are formed in each sporangium. In the sporangia, the spores are separated by a transverse septum. After release from the sporangial envelope, the spores become motile by means of peritrichous flagella.

Cultural Characteristics of Planobispora rosea ATCC 53773

For the examination of the cultural characteristics, Planobispora rosea ATCC 53773, was cultivated on various standard media suggested by Shirling and Gottlieb (Shirling E. B. and Gottlieb D., 1966--Method for characterization of Streptomyces species--Int. J. Syst. Bacteriol, 16, 313-340) with the addition of several media recommended by Waksman (Waksman, S. A. 1961--The Actinomycetes--The Williams and Wilkins Co. Baltimore; Vol. 2, 328-334).

Color determination was made, when necessary, by the method of Maerz and Paul (Maerz A. and M. Rea Paul, 1950--A Dictionary of Color--2nd Edition McGraw-Hill Book Company Inc. New York).

The ability of the organism to utilize different carbon sources was investigated by the method described by Shirling and Gottlieb.

The cultural and physiological characteristics and the carbon sources utilization are reported in Tables I, II, III.

The readings in Table I have been taken after two weeks of incubation at 28° C.

                  TABLE I                                                          ______________________________________                                         Cultural characteristics of strain                                             Planobispora rosea ATCC 53773                                                  Culture media                                                                               Morphological Characteristics                                     ______________________________________                                         Oatmeal agar 6%                                                                             Abundant growth with smooth surface, coral                                     pink (2-H-10) abundant production of light                                     pink aerial mycelium (1-A-9)                                      ISP 2 (yeast extract                                                                        Abundant growth with wrinkled surface, light                      malt agar)   pink (2-E-9), trace of light aerial mycelium                      ISP 3 (oatmeal agar 2%)                                                                     Moderate growth with smooth surface, light                                     pink (2-E-8), trace of pinkish white aerial                                    mycelium                                                          ISP 4 (inorganic salts                                                                      Moderate growth with smooth surface, coral                        starch agar) pink (2-E-10)                                                     ISP n 5 (glyercol                                                                           Moderate growth with smooth and flat surface,                     asparagine agar)                                                                            light pink (2-A-9), abundant production of                                     white aerial mycelim                                              ISP 6 (peptone yeast                                                                        Moderate growth, slightly wrinkled light coral                    extract iron agar)                                                                          pink (1-A-10)                                                     ISP 7 (tyrosine agar)                                                                       Moderate growth with smooth and thin surface                                   light pink (1-A-9), abundant formation of light                                pink (1-C-9) aerial mycelium                                      Hichey and Tresner's                                                                        Abundant growth with thick and wrinkled                           agar         surface light coral pink (1-A-10), moderate                                    production of light pink aerial mycelium                          Czapek glucose agar                                                                         Very scarce growth with smooth and thin                                        surface, moderate production of light pink                                     aerial mycelium                                                   Glucose asparagine                                                                          Moderate growth with smooth and thin surface                      agar         colorless, aerial mycelium absent                                 Nutrient agar                                                                               Good growth with smooth surface light orange                                   with a pinkish tinge (9-A-7)                                      Bennett's agar                                                                              Moderate growth with slightly wrinkled surface                                 light amber pink (10-A-6)                                         Calcium malate agar                                                                         Poor growth with smooth and flat                                               surface colorless                                                 Skim milk agar                                                                              Moderate growth with smooth surface coral                                      pink (2-F-9)                                                      Egg albumin agar                                                                            Poor growth with smooth and thin surface                                       colorless to light ink (2-A-8)                                    Dextrose tryptose                                                                           No growth                                                         agar                                                                           Potato agar  Good growth with smooth surface light orange                                   with a pinkish tinge (9-A-7)                                      ______________________________________                                    

Letter and numbers refer to the color determined according to Maerz and Paul (see above)

Physiological Characteristics of Planobispora rosea ATCC 53773

                  TABLE II                                                         ______________________________________                                         Tests                 Results                                                  ______________________________________                                         Starch hydrolysis     positive                                                 Hydrogen sulphide formation                                                                          negative                                                 Tyrosine reaction     positive                                                 Casein hydrolysis     weakly positive                                          Calcium malate digestion                                                                             negative                                                 Gelatin liquefaction  weakly positive                                          Milk coagulation      negative                                                 Milk peptonization    negative                                                 Nitrate reduction     positive                                                 ______________________________________                                    

                  TABLE III                                                        ______________________________________                                         Carbohydrate utilization                                                              Carbon source                                                                           Growth                                                         ______________________________________                                                Arabinose                                                                               +                                                                     Xylose   +                                                                     Ribose   -                                                                     Fructose +/-                                                                   Galactose                                                                               -                                                                     Glucose  +                                                                     Rhamnose -                                                                     Lactose  -                                                                     Sucrose  -                                                                     Maltose  +                                                                     Raffinose                                                                               -                                                                     Cellulose                                                                               -                                                                     Mannitol -                                                                     Salicin  +                                                                     Inositol +                                                                     Cellobiose                                                                              -                                                              ______________________________________                                          + moderate growth                                                              +/- scarce growth                                                              - no growth                                                              

For this test medium No. 8 is employed and the results are evaluated after 2 weeks of incubation at 28°-30° C.

Sensitivity to Temperature

The optimum growth temperature ranges from 28° C. to 37° C. No growth is observed at 15° C. and 50° C., moderate growth at 20° C.

Chemotaxonomical Characteristics

Cell wall analysis:

The analysis of aminoacids present in the cell wall was carried out by the method described by Becker et al ("Rapid differentiation between Nocardia and Streptomyces by paper chromatography of whole cell hydrolyzates", Appl. Microbiol. 12, 421-423, 1964).

The analysis of the whole cell hydrolyzates revealed the presence of meso-diaminopimelic acid.

No glycine was found upon analysis of the pure cell wall preparation obtained according to the method of Kawamoto et al. (Kawamoto I., O. Tetsuo, and N. Takashi, "Cell wall composition of Micromonospora olivoasterosporia, Hicromonospora sagamiensis and related organism. J. of Bacteriol. 146, 527-534, 1981).

Whole cell sugar pattern:

The analysis of the sugar content in the whole cell hydrolyzates was carried out by the method of Lechevalier M. P. ("Identification of aerobe actinomycetes of clinical importance" J. Lab. Clin. Med. 71, 934-944, 1968), using thin layer chromatography cellulose sheets as described by Staneck J. L. and G. D. Roberts ("Simplified approach to identification of aerobic actinomycetes by thin layer chromatography", Appl. Microbiol. 28, 226-231, 1974) with the following solvent system: Ethylacetate-Pyridine-Water (100:35:25 v/v).

The obtained results showed the presence of madurose (3-O-methyl-D-galactose) and absence of arabinose and galactose.

Identity of Strain Planobispora rosea ATCC 53773

This strain was assigned to the genus Planobispora and classified as Planobispora rosea because of the following morphological and chemical characteristics:

a) The presence of meso-diaminopimelic acid and the absence of glycine in the cell wall (cell wall chemotype III)

b) The presence of madurose in the whole cell hydrolyzate (whole cell sugar pattern B)

c) The formation on the aerial mycelium of long and cylindrical sporangia containing a pair of motile spores

d) The pink color of the vegetative mycelium.

The morphological characteristics of Planobispora rosea ATCC 53773 reported above are not substantially different from those of a strain of Planobispora rosea which was described by J. E. Thiemann et al in "The Actinomycetales", The Jena Intern. Symp. on Taxon., September 1968, ed. H. Prauser, Jena. It was deposited with the American Type Culture Collection where it received accession number 23866. No antibiotic production was described for this strain.

As with other microorganisms, the characteristics of the GE 2270 producing strains are subject to variation. For example, artificial variants and mutants of the strain can be obtained by treatment with various known mutagens, such as U.V. rays, X-rays, high frequency waves, radioactive rays, and chemicals such as nitrous acid, N-methyl-N'-nitro-N-nitrosoguanidine, and many others. All natural and artificial variants and mutants which belong to a species of the genus Planobispora and produce antibiotic GE 2270 are deemed equivalent to strain Planobispora rosea ATCC 53773 for the purposes of this invention.

As mentioned above, antibiotic GE 2270 B₁, B₂, C₁, C₂, D₁, D₂, E and T, are generally found mainly in the mycelium of the producing strain, while a minor amount of substance is found also in the fermentation broth.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the H-N.M.R. Spectrum of antibiotic GE 2270 Factor E.

FIG. 2 shows the H-N.M.R. Spectrum of antibiotic GE 2270 Factor T.

RECOVERY AND ISOLATION OF THE ANTIBIOTIC SUBSTANCES OF THE INVENTION

The recovery of antibiotic GE 2270 B₁, B₂, C₁, C₂, D₁, D₂, E and T from the mycelium or the fermentation broths of the producing microorganism is conducted according to known per se techniques such as extraction with solvents, precipitation by adding non-solvents or by changing the pH of the solution, partition chromatography, adsorption chromatography, reverse-phase partition chromatography, ion-exchange chromatography, molecular exclusion chromatography and the like.

A preferred procedure for recovering the antibiotic substances of the invention from the mycelium includes extracting the filtered or centrifugated mycelium with a water-miscible organic solvent, concentrating the extracts and recovering the crude antibiotic substance by precipitation, optionally with the addition of a precipitating agent, by extraction of the aqueous residue with a water-immiscible organic solvent or by adsorption chromatography followed by elution of the desired product from the absorption matrix.

A preferred procedure for recovering the antibiotic substances of the invention from the fermentation broth includes extraction with a water-immiscible organic solvent, followed by precipitation from the concentrated extracts, possibly by adding a precipitating agent, or by further extraction of an aqueous residue thereof with a water-immiscible solvent. Alternatively, the fermentation broth can be contacted with an adsorption matrix followed by elution with a polar elution mixture. This chromatographic procedure can also be applied to a concentrated extract obtained from the fermentation broth instead of on the broth itself.

The term "water-miscible solvent" as used in this application, is intended to have the meaning currently given in the art to this term and refers to solvents that, at the conditions of use, are miscible with water in a reasonably wide concentration range.

Examples of water-miscible organic solvents that can be used in the extraction of the antibiotic substances of the invention from the mycelial mass are: lower alkanols, e.g. (C₁ -C₃)alkanols such as methanol, ethanol and propanol; phenyl(C₁ -C₃)alkanols such as benzyl alcohol; lower ketones, e.g. (C₃ -C₄)ketones such as acetone and ethylmethylketone; cyclic ethers such as dioxane and tetrahydrofurane; glycols and their products of partial etherification, such as ethylene glycol, propylene glycol and ethylene glycol monomethyl ether; lower amides such as dimethylformamide, diethylformamide; and dimethylsulfoxide.

The term "water-immiscible solvent" as used in this application, is intended to have the meaning currently given in the art to this term and refers to solvents that at the conditions of use are slightly miscible or practically immiscible with water in a reasonably wide concentration range, suitable for the intended use.

Examples of water-immiscible organic solvents that can be used in the extraction of the antibiotic substance of the invention from the fermentation broth are: the usual hydrocarbon solvents which may be linear, branched or cyclic such as hexane or cyclohexane; halogenated hydrocarbons such as chloroform, carbon tetrachloride, dichloroethane, fluorobromoethane, dibromoethane, trichloropropane, chlorotrifluorooctane and the like; aromatic hydrocarbons such as benzene, toluene, xylene and the like; esters of at least four carbon atoms, such as ethyl acetate, propyl acetate, ethyl butyrrate, and the like; alkanols of at least four carbon atoms which may be linear, branched or cyclic such as butanol, 1-pentanol, 2-pentanol, 3-pentanol, 1-hexanol, 2-hexanol, 3-hexanol, 3,3-dimethyl-1-butanol, 4-methyl-1-pentanol; 3-methyl-1-pentanol, 2,2-dimethyl-3-pentanol, 2,4-dimethyl-3-pentanol, 4,4-dimethyl-2-pentanol, 5-methyl-2-hexanol, 1-heptanol, 2-heptanol, 5-methyl-1-hexanol, 2-ethyl-1-hexanol, 2-methyl-3-hexanol, 1-octanol, 2-octanol, cyclopentanol, 2-cyclopentylethanol, 3-cyclopentyl-1-propanol, cyclohexanol, cycloheptanol, cyclooctanol, 2,3-dimethylcyclohexanol, 4-ethylcyclohexanol, cyclooctylmethanol, 6-methyl-5-hepten-2-ol, 1-nonanol, 2-nonanol, 1-decanol, 2-decanol and 3-decanol; straight or branched alkyl ethers and mixture thereof such as ethyl ether, propyl ether, butyl ether, etc; and mixtures or functional derivatives thereof.

As known in the art, product extraction may be improved by salting or by adding a proper organic salt forming a ion pair with the antibiotic which is soluble in the extraction solvent.

When following an extraction an aqueous phase is recovered containing a substantial amount of an organic solvent, it may be convenient to azeotropically distill water from it. Generally, this requires adding a solvent capable of forming minimum azeotropic mixtures with water, followed by the addition of a precipitating agent to precipitate the desired product, if necessary. Representative examples of organic solvents capable of forming minimum azeotropic mixtures with water are n-butanol, benzene, toluene, butyl ether, carbon tetrachloride, chloroform, cyclohexane, 2,5-dimethylfurane, hexane and m-xylene; the preferred solvent being n-butanol.

Examples of precipitating agents are petroleum ether, lower alkyl ethers, such as ethyl ether, propyl ether and butyl ether, and lower alkyl ketones such as acetone.

After recovery of the crude mixture as described above, it might be necessary to submit it to a further purification/concentration step before separating the single antibiotic substances of the invention. A chromatographic procedure is the first choice, in this case.

As mentioned above, the antibiotic factors of the invention are normally co-produced with antibiotic GE 2270 factor A and represent a minor fraction compared to it. Therefore, it is generally necessary to separate factor A from the remaining antimicrobially active fractions and to concentrate these "minor" fractions before separation in order to recover the single antibiotic substances of the invention in appreciable amounts.

Examples of chromatographic systems that can be conveniently used in the step mentioned above are polystyrene or mixed polystyrene-divinylbenzene resins such as Amberlite XAD2 or XAD4 (Rohm and Haas), Dowex S112 (Dow Chemical Co.) and Diaion HP 20 (Mitsubishi); acrylic resins such as XAD7 or XAD8 (Rohm and Haas); polyamides such as polycaprolactames, nylons and cross-linked polyvinylpyrrolidones generally having a pore volume (ml/g) ranging between 1 and 5, surface area (m² /g) ranging between 1 and 100, apparent density (g/ml) ranging between 0.15 and 0.50, average pore diameter (Angstrom units) ranging between 100 and 3000 and particles size distribution where at least 40 percent of the particle size is lower than 300 micrometers, such as Polyamide-CC 6, Polyamide-SC 6, Polyamide-CC 6.6, Polyamide-CC 6AC and Polyamide-SC 6AC (Macherey-Nagel & Co., West Germany), the polyvinylpyrrolidone resin PVP-CL (Aldrich Chemie GmbH & Co., KG, West Germany), the polyamide resin PA 400 (M. Woelm AG, West Germany); and carbon.

In the case of polystyrene or acrylic resin a preferred eluent is a polar solvent mixture of water-miscible solvents such as those reported above; in the case of a polyamide resin the eluent is preferably an aqueous mixture of a water-miscible solvent, such as the ones mentioned above, while for carbon a preferred eluent is a lower ketone such as acetone or a lower alcohol such as methanol.

Further chromatographic procedures that can be conveniently used in the step mentioned above include also chromatography on stationary phases such as silica gel, allumina, diatomaceous earth and the like, with an organic eluting phase made of solvents including halogenated hydrocarbons, lower alkanols, ethers, and higher ketones of the type already mentioned above and mixtures thereof.

Conveniently, also the so-called steric exclusion chromatography can be employed with good purification results. In particular, controlled pore cross-linked dextrans in which most of the hydroxyl groups have been alkylated, e.g. Sephadex LH-20 (Pharmacia Fine Chemicals, Ab), are usefully employed in this case.

According to the ordinary procedures described above, it is usually obtained a purified mixture which, beyond the invention compounds, may still contain a lower amount of antibiotic GE 2270 factor A.

Separation of antibiotic GE 2270 factors B₁, B₂, C₁, C₂, D₁, D₂, E and T from the purified mixture can be done in any of the art-known ways.

Particularly preferred for separation purposes are the chromatographic techniques, such as those mentioned above, while reverse-phase chromatography appears to be the preferred separation technique. In addition to conventional reverse-phase column chromatography, also preparative HPLC using a reverse-phase column is usefully employed.

The stationary phase in this chromatographic technique may be one of those usually employed such as silanized silica gel having various functional derivatizations and eluting with an aqueous mixture of water-miscible solvents of the kind mentioned above.

Examples of functionalized silanized silica gels are those bearing (C₈ -C₂₂)alkyl groups, such as those wherein the functionality is represented by e.g. octadecylsilane or octylsilane moieties, or cyclohexane, phenyl, and similar functions. These resins are commercially available and new additions are regularly registered having similar or even improved properties that can be usefully employed in the process of the invention.

A specifically preferred preparative HPLC technique employs an octadecyl functionalized silica gel and an eluting mixture containing acetonitrile, tetrahydrofuran and aqueous ammonium formate.

A specifically preferred elution mode is represented by an elution with a mixture of phase A and phase B of about 25:75, wherein phase A is a mixture of acetonitrile:tetrahydrofuran:40 mM ammonium formate, 40:40:20 and phase B is a mixture of the same components but in proportion 10:10:80.

Fractions are collected as usual according to their content, e.g. by following the elution profile with a conventional U.V. detector at 254 nm, the solvents are then removed according to known per se techniques (e.g. evaporation under reduced pressure, lyophilization etc.) to isolate the pure antibiotic factors of the invention which, optionally, may be crystallized from lower alkanols such as methanol, ethanol, propanol and isopropanol.

As usual in this art, the production as well as the recovery and purification steps may be monitored by a variety of analytical procedures including bioassays such as paper disc or agar diffusion assays on sensible microorganisms, TLC or HPLC procedures, which may involve a UV or microbial detection step.

A preferred HPLC analytical technique is represented by a reverse-phase HPLC using a column with porous and spheric particles of silanized silica gel, e.g. silica gel functionalized with C-8 alkyl groups having a uniform diameter (such as 5 micrometer Bakerbond® C8, Baker Research Products, U.S.A.) and an eluent which is a linear gradient mixture of a polar water miscible solvent, such as those described above with a gradient of increasing polarity.

In this case a preferred elution mixture is the following:

Phase A: CH₃ CN:tetrahydrofuran:40 mM HCOONH₄, 40:40:20

Phase B: CH₃ CN:tetrahydrofuran:40 mM HCOONH₄, 10:10:80,

while a preferred elution mode is represented by a linear gradient from 20% to 30% of phase A in phase B in about 20 min, with a flow rate of about 1.8 ml/min and UV detection at 254 nm.

Physico-Chemical Characteristics of Antibiotic GE 2270 Factors B₁, B₂, C₁, C₂, D₁, D₂, E and T

A) The ultraviolet absorption spectra recorded with a Perkin Elmer Model 320 spectrometer exhibit the following absorption maxima:

    ______________________________________                                                           λ max (nm)                                            ______________________________________                                         ANTIBIOTIC GE 2270 FACTORS, B.sub.1, B.sub.2, C.sub.1, C.sub.2, D.sub.1,       D.sub.2                                                                        AND E:                                                                         0.1 M HCl           245-250 (shoulder)                                                             300-315                                                    0.1 M KOH           245-250 (shoulder)                                                             300-315                                                    Phosphate buffer pH 7.38                                                                           245-250 (shoulder)                                                             300-315                                                    Methanol            245-250 (shoulder)                                                             300-315                                                    ANTIBIOTIC GE 2270 FACTOR T:                                                   0.1 M HCl           250                                                                            280 (shoulder)                                                                 345 (shoulder)                                             0.1 M KOH           240 (shoulder)                                                                 290 (shoulder)                                                                 345 (shoulder)                                             Phosphate buffer pH 7.38                                                                           250 (shoulder)                                                                 310 (shoulder)                                                                 345 (shoulder)                                             Methanol            240 (shoulder)                                                                 275 (shoulder)                                                                 290 (shoulder)                                                                 311                                                                            335 (shoulder)                                             ______________________________________                                    

B) The infrared absorption spectra in nujol mull (antibiotic GE 2270 factors D₁, D₂, E and T) exhibit the following absorption maxima ν(cm⁻¹):

Factor D₁ : 3700-3100; 3020-2750 (nujol); 1645; 1570-1490; 1460 and 1375 (nujol); 1305; 1260-1100; 1100-870; 840; 800; 760; 720 (nujol); 700

Factor D₂ : 3700-3100; 3020-2750 (nujol); 1645; 1570-1490; 1460 and 1375 (nujol); 1305; 1260-1100; 1100-870; 840; 800; 760; 720 (nujol); 700

Factor E: 3700-3100; 3020-2750 (nujol); 1645; 1570-1490; 1460 and 1375 (nujol); 1305; 1260; 1250-1070; 1015; 985-900; 840; 800; 760; 740; 720 (nujol); 700

Factor T: 3700-3100; 3100; 3020-2750 (nujol); 1655; 1570-1490; 1460 and 1375 (nujol); 1410; 1300; 1240; 1200-1000; 980; 930; 890; 840; 805; 765; 745; 720 (nujol); 700

C) The ¹ H-NMR spectra (antibiotic GE 2270 factors D₁, D₂, E and T) exhibit the following groups of signals with a Bruker spectrometer in DMSO-d₆ (hexadeuterodimethyl-sulfoxide) using TMS as the internal standard (0.00 ppm) δ, ppm, m! (s=singlet, br s=broad singlet, d=doublet, dd=doublet of doublets, t=triplet, m=multiplet, Py=pyridine, Tz=thiazole)

Factor D₁ (recorded at 500 MHz): 8.88, d, (NH); 8.70, d, (2NH's); 8.57, s, 8.50, s, 8.25, s, 8.21, s and 7.35, s, (CH's of 5 thiazoles); 8.40, m, (glycine NH); 8.28-8.21, m, (pyridine CH's); 7.32-7.20, m, (aromatic CH's and primary amide NH); 7.00, s, 6.84, s, 6.53, s, (primary amide NH's); 5.95, d, (OH); 5.29-5.15, m, (αCH's); 5.04, m, (phenylserine βCH); 4.81, m and 4.56, m, (oxazoline CH₂); 4.30-3.80, m, (CH₂ of glycine and prolineamide CH's); 2.72, m, and 1.43, m, (CH₂ of asparagine); 2.60, s, (CH₃); 2.21-1.91, (m), (isopropyl CH and prolineamide CH's); 0.90 (d) and 0.86 (d), (valine CH₃)

Factor D₂ (recorded at 500 MHz): 9.00, d, (NH); 8.69, br s (2NH's); 8.59, s, 8.53, s, 8.29, s and 7.35, s, (thiazole CH's); 8.38, m, (glycine NH); 8.40 and 8.26 (m), (Py.CH's); 7.37-7.18, m, (aromatic CH's, primary amide NH); 6.97, s, (primary amide NH); 6.03, d and t, (2 OH's); 5.28-5.16, m (αCH's); 5.03, m, (βCH); 4.97, m, CH₂ (OH)!; 4.79 and 4.55, m (CH₂ of oxazoline); 3.97-3.76, m, (CH₂ of glycine and CH's of prolineamide); 2.71, m and 1.28, m, (CH₂ of N-methylasparagine); 2.18-1.89, m, (isopropyl CH and prolineamide CH₂ 's); 0.88, d and 0.84 d (valine CH₃ 's)

Factor E (recorded at 500 MHz): 8.95, d, (NH); 8.73, d, (NH); 8.60, s, (Tz.CH); 8.57, d, (NH); 8.53, s, (Tz.CH); 8.42, m, (Py.CH's); 8.31, m, (NH); 8.28, m, (Py.CH); 8.24, s, (Tz.CH); 7.33, s, (Tz.CH); 7.31-7.20, m, (aromatic CH's, primary amide NH); 6.98, s, 6.91, s, 6.62, s, (primary amide NH's); 6.04, d, (OH); 5.95, t, (OH); 5.28-5.14, m (αCH's), 5.03, m, (βCH); 4.99, m, CH₂ (OH)!; 4.81, dd and 4.57, dd, (oxazoline CH₂); 4.26 (m) and 3.79 (m), (glycine CH₂); 4.25, m, 3.98, m, 3.82, m, (prolineamide CH's); 2.77, m, and 1.25, m, (CH₂ of asparagine); 2.60, s, (CH₃); 2.20, m, and 1.89, m, (valine βCH and CH₂ of prolineamide); 0.90, d, 0.84, d, (valine CH₃ 's)

Factor T (recorded at 250 MHz): 8.95, d, (NH); 8.70, d, (2NH's); 8.66, s, 8.65, s, 8.60, s, 8.30, s and 7.38, s, (CH's of 4 thiazoles and 1 oxazole); 7.35-7.24, m, (aromatic CH's and primary amide NH); 6.68 (primary amide NH); 5.96, d, (OH); 5.34-5.18, m (αCH's); 5.05, m, (βCH); 5.03, s, CH₂ (OCH₃)!; 4.32, m and 3.82, m, (CH₂ of glycine); 4.48, m, 4.04, m and 3.63, m, (CH's of prolineamide); 3.40, s, (OCH₃); 2.73, m and 1.41, m, (CH₂ of N-methylasparagine); 2.60, B, (CH₃); 2.49, d, (CH₃ of N-methylasparagine); 2.27-1.88, m (isopropyl CH and CH₂ of prolineamide); 0.89, d, (CH₃ of valine)

FIGS. 1 and 2 show the ¹ H-NMR spectra of antibiotic GE 2270 factor E and factor T, respectively.

D) The retention times (R_(t)) (antibiotic GE 2270 factors B₁, B₂, C₁, C₂, D₁, D₂, E and T) in the following reverse phase HPLC system:

Column: Bakerbond® C8 (5 μm) 4.6×250 mm (Bakerbond is a trade name for reverse phase octylsilyl silica gel HPLC columns supplied by J. T. Baker Research Product, Phillisburg, N.J. 08865 USA)

Flow rate: 1.8 ml/min

Phase A: CH₃ CN:tetrahydrofuran:40 mM HCOONH₄ 40:40:20

Phase B: CH₃ CN:tetrahydrofuran:40 mM HCOONH₄ 10:10:80

Elution: linear gradient from 20% to 30% of Phase A in 20 min

Detection: UV 254 nm

are as reported below:

    ______________________________________                                                         Retention time                                                                 relative to                                                    Antibiotic      factor A   R.sub.t                                             GE 2270 factor  (R.sub.t 16.6 min)                                                                        (min)                                               ______________________________________                                         E               0.52       8.62                                                D.sub.1         0.56       9.36                                                D.sub.2         0.59       9.72                                                C.sub.1         0.65       10.78                                               C.sub.2         0.76       12.60                                               B.sub.1         0.85       14.18                                               B.sub.2         0.97       16.08                                               T               1.19       19.68                                               ______________________________________                                    

E) The main FAB-MS peaks obtained on a Kratos MS-50 double focusing mass spectrometer, using 8 kV accelerating voltage and a saddle field atom gun with Xe gas (2×10⁻⁵ torr pressure indicated on the source ion guage) at 6 kV voltage and 1 mA current, the sample being mixed with a thioglycerol matrix containing 0.1M acetic acid! are as indicated below. The values correspond most likely to the lowest isotope of the protonated molecular ion:

    ______________________________________                                         Antibiotic     FAS-MS                                                          GE 2270 factor M + 1 ion                                                       ______________________________________                                         E              1262                                                            D.sub.1        1232                                                            D.sub.2        1276                                                            C.sub.1        1246                                                            C.sub.2        1276                                                            B.sub.1        1276                                                            B.sub.2        1260                                                            T              1288                                                            ______________________________________                                    

On the basis of the physico-chemical data reported above the following structure formulas can be tentatively assigned to antibiotic GE 2270 factors D₁, D₂, E and T: ##STR1##

The antimicrobial activity of the compounds of the invention can be demonstrated by a series of standard tests in vitro.

Minimal inhibitory concentrations (MIC) were determined by microbroth dilution methodology. Inocula were 10⁴ -10⁵ CFU per ml. All microorganism were cultured at 37° C. MIC were read at 18-24 h, except for Neisseria gonorrhoeae, B. fragilis, and P. acnes (48 h). N. gonorrhoeae was incubated in a 5% CO₂ atmosphere; anaerobes were incubated in an anaerobic gas mixture. Media used were: Oxoid Iso-Sensitest broth for staphylococci, Enterococcus faecalis, Escherichia coli, Proteus vulgaris, and Pseudomonas aeruginosa; Difco Todd-Hewitt broth for other streptococci; Difco GC base broth with 1% BBL IsoVitalex for N. gonorrhoeae; Difco Wilkins-Chalgren broth for P. acnes and B. fragilis (10⁵ CFU/ml).

The minimal inhibitory concentrations (MIC, microgram/ml) of the antibiotic factors of the invention are reported below in Table IV.

                  TABLE IV                                                         ______________________________________                                                    ANTIBIOTIC GE 2270 FACTORS (μg/ml)                               STRAIN       B.sub.1  B.sub.2  C.sub.1                                                                               C.sub.2                                  ______________________________________                                         Staph. aureus L165 Tour                                                                     1        1        1      1                                        Staph. epidermidis                                                                          1        0.25     0.25   1                                        L147 ATCC 12228                                                                Staph. haemolyticus                                                                         4        2        1      2                                        L602                                                                           Strep. pyogenes L49                                                                         0.25     1        0.25   1                                        C203                                                                           Strep. pneumoniae                                                                           0.5      0.13     0.13   0.25                                     L44 UC41                                                                       Enterococcus faecalis                                                                       0.5      1        0.25   1                                        L149 ATCC 7080                                                                 Prop. acnes L1014                                                                           0.016    0.016    0.008  0.008                                    ATCC 6919                                                                      Bact. fragilis L1010                                                                        4        32       1      8                                        ATCC 23745                                                                     Neiss. gonorrhoeae                                                                          >128     >128     >128   128                                      L997 ISM 68/126                                                                Esch. coli L47                                                                              >128     >128     >128   >128                                     SKF 12140                                                                      Pseud. aeruginosa                                                                           >128     >128     >128   >128                                     ATCC 10145                                                                     Prot. vulgaris ATCC 881                                                                     >128     >128     >128   >128                                     ______________________________________                                                    ANTIBIOTIC GE 2270 FACTORS (μg/ml)                               STRAIN       D.sub.1  D.sub.2  E      T                                        ______________________________________                                         Staph. aureus L165 Tour                                                                     2        1        2      0.25                                     Staph. epidermidis                                                                          0.5      0.5      2      0.25                                     L147 ATCC 12228                                                                Staph. haemolyticus                                                                         8        4        >128   1                                        L602                                                                           Strep. pyogenes L49                                                                         0.25     0.5      0.5    1                                        C203                                                                           Strep. pneumoniae                                                                           0.13     0.13     0.13   0.25                                     L44 UC41                                                                       Enterococcus faecalis                                                                       0.5      1        1      0.13                                     L149 ATCC 7080                                                                 Prop. acnes L1014                                                                           0.016    0.016    0.03   0.06                                     ATCC 6919                                                                      Bact. fragilis L1010                                                                        >128     64       128    8                                        ATCC 23745                                                                     Neiss. gonorrhoeae                                                                          >128     >128     >128   8                                        L997 ISM 68/126                                                                Esch. coli L47                                                                              >128     >128     >128   >128                                     SKF 12140                                                                      Pseud. aeruginosa                                                                           >128     >128     >128   >128                                     ATCC 10145                                                                     Prot. vulgaris ATCC 881                                                                     >128     >128     >128   >128                                     ______________________________________                                    

In view of their properties, the compounds of the invention can be used as active ingredients in the preparation of medicaments for human or animal treatment.

In particular, antibiotic GE 2270 factor B₁, B₂, C₁, C₂, D₁, D₂, E or T is an antimicrobial agent mainly active against gram positive bacteria and gram positive as well as gram negative anaerobes. It appears to be very active also in Staphylococcal endocarditis without any cross-resistance with meticillin, aminoglycosides or glycopeptide antibiotics.

The main therapeutic indication of the antibiotic substance of the invention is thus in the treatment of infections related to the presence of a microorganism susceptible to it.

The term "treatment" is intended to encompass also prophylaxis, therapy and cure.

The patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.

The compounds of the invention can be administered as such or in admixture with pharmaceutically acceptable carriers and can also be administered in conjunction with other antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and glycopeptides. Conjunctive therapy, thus includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutical effects of the first administered one is not entirely disappeared when the subsequent is administered.

A preferred pharmaceutical formulation is represented by a formulation suitable for a topical application on an intact or damaged skin or mucous membrane. Examples of such formulations are powders, ointments, creams and lotions. The excipients in these formulations are the usual pharmaceutically acceptable vehicles such oleaginous ointment bases (e.g. cetyl esters wax, oleic acid, olive oil, paraffin, spermaceti, starch glycerite); absorbent ointment bases (e.g. anhydrous lanolin, hydrophilic petrolatum), emulsion ointment bases (e.g. cetyl alcohol, glyceryl monostearate, lanolin, stearic acid), water-soluble ointment bases (e.g. glycol ethers and their derivatives which include polyethylene glycols, poly(oxy-1,2-ethanediyl)-alpha-hydro-omega-hydroxy-octadecanoate, polysorbates, and polyethylene glycols mono-stearates).

These formulations may contain other known excipients, such as preservatives and are prepared as known in the art and reported in reference handbooks such as Remington's Pharmaceutical Sciences, Seventeenth edition, 1985, Mack Publishing Co.

The compounds of the invention can also be formulated into formulations suitable for parenteral administration according to precedures known per se in the art and reported in reference books such as the one mentioned above.

For instance, a compound of the invention is formulated with a solubilizing agent such as polypropylene glycol or dimethylacetamide and a surface-active agent such as polyoxyethylene sorbitan mono-oleate or polyethoxylated castor oil in steril water for injection.

An example of a typical formulation for parenteral administration contains 10 mg of antibiotic GE 2270 factor B₁, B₂, C₁, C₂, D₁, D₂, E or T for ml of final preparation, 10-20% of a surface-active agent which may be a polyoxyethylene sorbitan fatty acid ester, a polyoxyethylene castor oil derivative or a polyoxyethylene hydrogenated castor oil derivative and 0-20%, and preferably 10-20% of a solubilizing agent such as propylene glycol, dimethylacetamide, dimethylformamide, ter-butyl-N-hydroxycarmabate, 1,2-, 1,3-, or 1,4-butandiol, ethyl oleate, tetrahydrofurfuryl-polyethylene-glycol 200, dimethyl isosorbide, benzyl alcohol and the like. A preferred solubilizing agent is propylene glycol.

Polyoxyethylene sorbitan fatty acid esters are commercially available and some of them are traded under the trade name "Tween". They are also known with the non-proprietary name of "polysorbates". Examples of them are polysorbate 20, 21, 40, 60, 61, 65, 80, 81 and 85. Preferred for use in the formulations of the invention is polysorbate 80 (sorbitan mono-9-octadecenoate, poly(oxy-1,2-ethanediyl)derivatives).

Polyoxyethylene castor oils and polyoxyethylene hydrogenated castor oils are also commercially available. Some of them are traded with the trade name "Cremophor". Examples of such compounds are those known as Cremophor EL (polyethoxylated castor oil), Cremophor RH 40 (polyethoxylated hydrogenated castor oil), Cremophor RH 60 (PEG 60 hydrogenated castor oil) or Emulphor EL-719 (polyoxyethylated vegetable oil).

Preferably, a formulation for injection should have a pH in the range of 7±0.5. If necessary, it might be advisable to adjust the pH of the preparation with a suitable buffering agent. Conveniently, TRIS (i.e. trihydroxymethylaminomethane) or phosphate can be used as buffering agents.

A preferred formulation for parenteral administration includes the following excipients: Cremophor® EL (polyoxyl 35 castor oil USP/NF) 20%, propylene glycol from 5 to 20%, preferably 10-20%.

Generally, these formulations can be prepared by dissolving the active ingredient into the organic solvent, then adding the surface active ingredient, and finally diluting to the desired volume with sterile water for injection.

Other excipients, such as preservative or stabilizing agents can be added as known in the art.

An example of a parenteral formulation is the following:

    ______________________________________                                         antibiotic GE 2270 factor T                                                                         10        mg                                              PEG 40 castor oil (Cremophor EL)                                                                    0.2       ml                                              propylene glycol     0.2       ml                                              methyl parahydroxybenzoate                                                                          0.5       mg                                              propyl parahydroxybenzoate                                                                          0.05      mg                                              water for injection q.s.                                                                            1         ml                                              ______________________________________                                    

In the above formulation, instead of 10 mg of antibiotic GE 2270 factor T, 10 mg of antibiotic GE 2270 factor B₁, B₂, C₁, C₂, D₁, D₂, or E can be used.

Alternatively, the active ingredient may be prepared as a lyophilized powder for reconstitution before use.

If the lyophilized material is prepared starting from a mixture containing the active ingredient and the surfactant, such as polyethylene glycol 60 hydrogenated castor oil, it can conveniently be reconstituted with the aqueous medium alone, without addition of an organic solvent.

Optionally, a common lyophilization aid can be added, if necessary, to obtain a lyophilized material in powder form.

Preferably, all these formulations are used for i.v. administration in the treatment of any infection involving a microorganism susceptible to the antibiotic of the invention.

In the treatment of pseudomembranous colitis or other diseases attributable to the presence of anaerobes in the gastrointestinal tract, an effective dose of the compound of the invention may be administered orally in a suitable pharmaceutical form such as a capsule, a tablet or an aqueous suspension.

The dosage of the active ingredient depends on many factors which include type, age and conditions of the patient, specific active ingredient and formulation selected for the administration, administration schedule, etc.

In general, effective antimicrobial dosages are employed per single unit dosage form.

Repeated applications/administrations, e.g. from 2 to 6 times a day, are in general preferred. An effective dosage may be in general in the range 0.5-50 mg/kg body weight/day.

A preferred topic preparation is an ointment containing from 1% to 10% of a compound of the present invention.

Anyway, the prescribing physician will be able to determine the optimal dosage for a given patient in a given situation.

Besides their use as medicaments in human and veterinary therapy, the compounds of the invention can also be used as animal growth promoters.

For this purpose, a compound of the invention is administered orally in a suitable feed. The exact concentration employed is that which is required to provide for the active agent in a growth promotant effective amount when normal amounts of feed are consumed.

The addition of the active compound of the invention to animal feed is preferably accomplished by preparing an appropriate feed premix containing the active compound in an effective amount and incorporating the premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containing the active ingredient can be blended into the feed. The way in which such feed premixes and complete rations can be prepared and administered are described in reference books (such as E. W. Crampton et al., "Applied Animal Nutrition", W. H. Freedman and CO., S. Francisco, USA, 1969 or D. C. Church, "Livestock Feeds and Feeding", O and B Books, Corvallis, Oreg., USA, 1977).

The following examples further illustrate the invention and have not to be interpreted as limiting it in any way.

EXAMPLE 1 Production of Antibiotic GE 2270

A culture of Planobispora rosea ATCC 53773 is grown on an oatmeal agar slant for two weeks at 28°-30° C. and then used to inoculate 500 ml flasks containing 100 ml of a seed medium of the following composition:

    ______________________________________                                         Starch           20          g/l                                               Polypeptone      5           g/l                                               Yeast extract    3           g/l                                               Beef extract     2           g/l                                               Soybean meal     2           g/l                                               Calcium carbonate                                                                               1           g/l                                               Distilled water q.s.                                                                            100         ml                                                (adjusted to pH 7.0 before sterilization)                                      ______________________________________                                    

The flask is incubated on a rotary shaker (200 rpm at 28°-30° C. for 92 h. The obtained culture is then used to inoculate a jar fermenter containing 4 liters of the same medium and the culture is incubated at 28°-30° C. for 48 hours with stirring (about 900 rpm) and aeration (about one standard liter of air per volume per minute).

The obtained broth is transferred to a fermenter containing 50 l of the following production medium:

    ______________________________________                                         Starch           20          g/l                                               Peptone          2.5         g/l                                               Hydrolyzed casein                                                                               2.5         g/l                                               Yeast extract    3           g/l                                               Beef extract     2           g/l                                               Soybean meal     2           g/l                                               Calcium carbonate                                                                               1           g/l                                               Distilled water  q.s.                                                          (adjusted to pH 7.0 before sterilization)                                      ______________________________________                                    

and incubated for about 72 hours at 28°-30° C.

Antibiotic production is monitored by paper disc agar assay using B. subtilis ATCC 6633 grown on minimum Davis medium. The inhibition zones are evaluated after incubation overnight at 35° C.

EXAMPLE 2 a) Recovery of Crude Antibiotic GE 2270

The fermentation mass (50 l) obtained above is harvested and submitted to filtration on a filter aid (Clarcell).

The mycelium is extracted twice with 20 l of methanol and the pooled extracts are concentrated under reduced pressure to give an aqueous residue which is extracted twice with ethyl acetate. Crude antibiotic GE 2270 (6.06 g) is precipitated by adding petroleum ether to the concentrated organic phase.

b) Isolation of a Crude Mixture of Antibiotic GE 2270 Factors

The crude obtained from the mycelium according to the procedure described above (3 g) is dissolved in tetrahydrofuran and concentrated under reduced pressure in the presence of silica gel (230-400 mesh). The obtained solid residue is collected and applied to a chromatography column containing 300 g of silica gel (230-400 mesh) prepared in methylene chloride (CH₂ Cl₂). The column is developed first with methylene chloride (2 l) and then sequentially with 1.5 l mixtures of methylene chloride and methanol in the following ratios: 98/2; 96/4, 94/6, 92/8, 90/10 and 88/12 (v/v).

Fractions are collected, analyzed by TLC, HPLC or microbiologically against B. subtilis and pooled according to their antibiotic content.

The pooled fractions enriched in antibiotic GE 2270 factors B₁, B₂, C₁, C₂, D₁, D₂, and E, and containing also a certain amount of antibiotic GE 2270 factor A, are concentrated under reduced pressure to give an oily residue from which a mixture of the above named antibiotic factors (1.15 g) is precipitated with petroleum ether.

c) Separation and Isolation of Antibiotic GE 2270 Factors B₁, B₂, C₁, C₂, D₁, D₂, and E

Antibiotic GE 2270 factors B₁, B₂, C₁, C₂, D₁, D₂ and E are separated and purified from the above obtained crude mixture by preparative HPLC using a 250×20 mm column packed with Nucleosil® C18 (silica gel functionalized with octadecylsilane groups) (5 μm) and eluted with mixtures of Phase A: CH₃ CN:tetrahydrofuran:40 mM HCOONH₄ (40:40:20); Phase B: CH₃ CN:tetrahydrofuran:40 mM HCOONH₄ (10:10:80). The antibiotic mixture (6 mg) was solubilized in 3 ml of Phase B and 1 ml of Phase A and was injected into the HPLC column which was eluted at a flow rate of 14 ml/min with a 26:74 mixture of Phase A and B. The eluted fractions were collected according to the UV adsorption profile at 254 nm. The fractions of subsequent chromatographic runs having homogeneous content were pooled and concentrated under reduced pressure to eliminate CH₃ CN. The residual solution showed antibacterial activity against Staphylococcus aureus Tour L165 by paper disc assay. These solutions were lyophilized at least three times to remove completely the HCOONH₄ buffer residue from the HPLC phases.

The yields were as follows: antibiotic GE 2270 factor E, 11 mg; antibiotic GE 2270 factor D₁, 12 mg; antibiotic GE 2270 factor D₂, 10 mg; antibiotic GE 2270 factor C₁, 2 mg; antibiotic GE 2270 factor C₂, 3 mg; antibiotic GE 2270 factor B₁, 2 mg; antibiotic GE 2270 factor B₂, 2 mg.

EXAMPLE 3 Production of Antibiotic GE 2270 Factor T a) Strain Fermentation

A culture of Planobispora rosea ATCC 53773 grown on agar slant was inoculated into two 500 ml Erlenmeyer flasks containing 100 ml of seed medium (starch 2%, polipeptone 0.5%, yeast extract 0.3%, beef extract 0.2%, soybean meal 0.2%, calcium carbonate 0.1%, brought to pH 7.0 before sterilization). Three 500 ml Erlenmeyer flasks of seed medium were inoculated (5% inoculum) with the culture incubated at 28° C. for 96 hours on a rotary shaker (200 rpm).

The cultures were incubated at 28° C. for 72 hours on a rotary shaker (200 rpm) and then were inoculated into a 10 l jar-fermentor containing 6 l of the seed medium. After 72 hours incubation at 28° C. with 1 l/V/min air flow and stirring at 900 rpm, the culture was inoculated into a jar-fermentor containing 200 l of production medium (starch 2%, peptone 0.25%, hydrolyzed casein 0.25%, yeast extract 0.3%, beef extract 0.2%, soybean meal 0.2%, calcium carbonate 0.1%, adjusted to pH 7.4 before sterilization).

Isolation of Antibiotic GE 2270 Factor T

After 126 hours of fermentation the broth was harvested and the mycelium was collected by filtration with Hyflo filter aid. The mycelium cake was extracted subsequently with 60 and 20 l of acetone and the pooled extracts were concentrated under reduced pressure. The crude antibiotic complex was separated from the water residue by centrifugation in a liquid-solid separator. The wet material was solubilized into 2-propanol and the solution was concentrated under reduced pressure to remove water. The crude antibiotic complex (50 g) precipitated from the concentrated residue. This crude complex contains a major quantity of antibiotic GE 2270 factor A along with antibiotic GE 2270 factor T and the other minor factors mentioned above.

For the separation of factor T from the other components of said crude complex, the preparations from 6 repeated fermentations were pooled and solubilized into 12 l of CH₂ Cl₂ :methanol (93:7). The insoluble material was removed by filtration and the solution, containing the antibiotic complex, was applied to a 13 kg (230-400 mesh) silica gel column equilibrated in CH₂ Cl₂ :methanol (93:7). Antibiotic GE 2270 factor T was eluted from the column by eluting with CH₂ Cl₂ :methanol (93:7). The fractions containing it (HPLC analysis) were pooled, concentrated under reduced pressure and dried to yield 8 g of antibiotic GE 2270 factor T together with minor impurities.

15 Mg of this crude substance was solubilized in 0.2 ml of dimethylformamide:CH₃ CN:water (50:25:25) and was injected into the HPLC system having a Hibar (E. Merck; Darmstadt F.R. Germany) 250×10 column packed with Lichrosorb® C18 (octadecyl silanized silica gel) (7 μm) and eluted with CH₃ CN:H₂ O (60:40) at a flow rate of 4.5 ml/min. Those fractions containing antibiotic GE 2270 factor T (U.V. detection at 254 nm) were then collected.

The fractions of this type coming from 15 subsequent chromatographic runs were pooled, concentrated under reduced pressure and dried to yield 150 mg of purified antibiotic GE 2270 factor T.

Preparation of the Analytical HPLC Reference Compound a) Production of Antibiotic GE 2270

A culture of Planobispora rosea ATCC 53773 is grown as described in Example 1.

b) Recovery of Antibiotic GE 2270

The fermentation mass (50 l) obtained above is harvested and submitted to filtration in the presence of a filter aid (Clarcell).

Antibiotic GE 2270 is found mainly in the mycelium, even if a certain amount of it can be recovered also from the filtrates.

a) The filtrate is adjusted to about pH 7.0 and extracted with ethyl acetate (50 l). The organic phase is separated by centrifugation and concentrated to a small volume under reduced pressure. The obtained oily residue is then treated with petroleum ether to precipitate crude antibiotic GE 2270 that is collected by filtration and dried. 415 mg of crude antibiotic GE 2270 complex is obtained.

b) The mycelium is extracted twice with 20 l of methanol and the pooled extracts are concentrated under reduced pressure to give an aqueous residue which is extracted twice with ethyl acetate. Crude antibiotic GE 2270 (6.06 g) is precipitated by addition of petroleum ether from the concentrated organic phase.

c) Purification of Antibiotic GE 2270 Factor A

The crude obtained from the mycelium according to the procedure described above (3 g) is dissolved in tetrahydrofuran and concentrated under reduced pressure in the presence of silica gel (230-400 mesh). The obtained solid residue is collected and applied to a chromatography column containing 300 g of silica gel (230-400 mesh) prepared in methylene chloride (CH₂ Cl₂). The column is developed first with methylene chloride (2 l) and then sequentially with 1.5 l mixtures of methylene chloride and methanol in the following ratios: 98/2; 96/4, 94/6, 92/8, 90/10 and 88/12 (v/v).

Fractions are collected, analyzed by TLC, HPLC or microbiologically against B. subtilis and pooled according to their antibiotic content.

The pooled fractions containing pure antibiotic GE 2270 factor A (HPLC retention time 14.9 min, see the physico-chemical data, point E, below) are concentrated under reduced pressure to give an oily residue which is solubilized with tetrahydrofuran. From this solution, antibiotic GE 2270 factor A (600 mg) is precipitated by adding petroleum ether.

Another crop of antibiotic GE 2270 factor A is obtained from other fractions separated by the above described chromatographic system but which contain it in an impure form (HPLC). Also these fractions are pooled, concentrated and treated to obtain a solid as described above. This crude preparation of antibiotic GE 2270 factor A is purified by HPLC according to the following procedure:

A portion of this precipitate (6 mg) is dissolved in acetonitrile:water, 1:1 (v/v) and injected into a HPLC chromatographic system equipped with a silanized silica gel column (Lichrosorb RP 18, 7 micrometer, 250×10 mm, Merck, Darmstadt).

Elution is made with a linear gradient of a mixture of solution A and B from 50% to 85% of A in B, in 20 min, at a flow rate of about 4 ml/min. Solution A is a mixture of acetonitrile and 18 mM sodium phosphate buffer 70/30 (v/v), adjusted to pH 6, while solution B is a mixture of acetonitrile and 18 mM phosphate buffer, 10/90 (v/v), adjusted to pH 6.

The column is connected to a Perkin Elmer LC85 UV detector at 330 nm. The fractions of 11 subsequent chromatographic runs having homogeneous content are pooled and concentrated under reduced pressure to remove acetonitrile thus obtaining separated residual solutions containing antibiotic GE 2270 factor A. These solutions are extracted twice with an equal volume of ethyl acetate and the antibiotic product is obtained by precipitation from the concentrated organic phase by adding petroleum ether. Upon recovery by filtration and drying, 27 mg of antibiotic GE 2270 factor A are obtained.

Physico-Chemical Characteristics of Antibiotic GE 2270 Factor A

A) The ultraviolet absorption spectrum which exhibits the following absorption maxima:

    ______________________________________                                                       E.sub.1cm.sup.1%                                                                        λ max (nm)                                       ______________________________________                                         0.1 M HCl                  245 (shoulder)                                                                 310                                                 0.1 M KOH                  245 (shoulder)                                                                 313                                                 Phosphate buffer pH 7.4    245 (shoulder)                                                                 314                                                 Methanol        265        244 (shoulder)                                                                 310                                                 ______________________________________                                    

B) The infrared absorption spectrum in nujol mull exhibits the following absorption maxima (cm⁻¹): 3700-3060; 3060-2660 (nujol); 1650; 1590-1490; 1490-1420 (nujol); 1375 (nujol); 1310; 1245; 1210; 1165; 1090; 1060; 1020; 970; 930; 840, 810, 750, 720 (nujol), 700;

The main functional I.R. absorption bands of this spectrum can be attributed as:

    ______________________________________                                         ν, (cm.sup.-1)                                                                             Assignment                                                      ______________________________________                                         3600-3100      ν NH, ν OH                                                1650           amide I (ν C═O)                                          1545           heterocyclic ν C═C and ν C═N                      1525, 1495     amide II (δ NH)                                           1250, 1205     aromatic δ CH                                             870            heterocyclic γ CH                                         745, 700       aromatic γ CH                                             ______________________________________                                    

C) The ¹ H-NMR spectrum exhibits the following groups of signals (in ppm) at 500 MHz recorded in DMSO-d₆ (hexadeuterodimethylsulfoxide) using TMS as the internal standard (0.00 ppm); the number of protons for each signal is reported between parenthesis: 9.02 (1); 8.68 (1); 8.70 (1); 8.57 (1); 8.50 (1); 8.43 (1); 8.37 (1); 8.26 (1); 8.25 (1); 7.4-7.20 (9); 6.96 (2); 6.02 (1); 5.30-5.18 (3); 5.01 (1); 4.97 (2); 4.80 (1); 4.56 (1); 4.30 (1); 4.26 (1); 3.98 (1); 3.81 (1); 3.79 (1); 3.38 (3); 2.72 (1); 2.58 (3); 2.48 (3); 2.16 (1); 2.13 (1); 1.96 (2); 1.88 (1); 1.34 (1); 0.87 (3); 0.84 (3);

D) The ¹³ C-NMR spectrum exhibits the following groups of signals (ppm) at 125 MHz in DMSO-d₆ with TMS as the internal reference (0.00 ppm), Q means quaternary carbon atoms or C═O groups; 173.69, Q; 171.10, Q; 169.83, Q; 169.51, Q; 168.45, Q; 168.26, Q; 167.84, Q; 165.68, Q; 164.75, Q; 161.40, Q; 161.23, Q; 160.46, Q; 160.29, Q; 159.35, Q; 153.42, Q; 150.31, Q; 150.11, Q; 149.41, Q; 146.93, Q; 144.73, Q; 143.75, Q; 142.10, Q; 141.78, Q; 141.33, CH; 140.97, Q; 139.53, Q; 128.68, CH; 127.99, 2 CH!; 127.67, Q; 127.67, CH; 126.88, CH; 126.76, 2 CH!; 123.17, CH; 118.66, CH; 116.42, CH; 73.81, CH; 69.41, CH₂ ; 67.97, CH; 67.36, CH₂ ; 60.12, CH; 58.63, CH₃ ; 58.24, CH; 55.41, CH; 48.15, CH; 47.03, CH₂ ; 41.19, CH₂ ; 37.60, CH₂ ; 34.06, CH; 29.76, CH₂ ; 25.85, CH_(3;) 24.28, CH₂ ; 18.49, CH₃ ; 17.98, CH₃ ; 11.99, CH₃ ;

E) The retention-time (R_(t)) is 14.9 min when analyzed by reverse phase HPLC under the following conditions:

column: Ultrasphere ODS (reverse phase silanized silica gel; 5 micrometer) Altex (Beckman) 4.6 mm (i.d.)×250 mm

pre-column: Brownlee Labs RP 18 (octadecylsilane silica gel; 5 micrometer)

eluent A: acetonitrile:18 mM sodium phosphate 70:30 (v/v), adjusted to pH 7.0

eluent B: acetonitrile:18 mM sodium phosphate 10:90 (v/v), adjusted to pH 7.0

elution mode: linear gradient of eluent A in eluent B from 45% to 70% in 20 min

flow rate: 1.8 ml/min

U.V. detector: 254 nm

internal standard: Chloramphenicol (R_(t) =3.7 min)

When the retention-time is measured under the conditions described in paragraph D) above regarding the characterization of the invention compounds, its value is 16.6 min.

F) The elemental analysis, after the sample has been previously dried at about 140° C. under inert atmosphere, which indicates the following composition: carbon, hydrogen, nitrogen, sulfur;

G) The R_(f) value is 0.37 in the following chromatographic system: dichloromethane:methanol, 9:1 (v/v) using silica gel plates (silica gel 60F₂₅₄, Merck Co) Visualization: U.V. light at 254 nm, yellow spot with iodine vapors or bioautography using B. subtilis ATCC 6633 on minimal Davis medium; internal standard: chloramphenicol (Rf 0.56)

H) The FAB-MS analysis shows the lowest mass isotope of the protonated molecular ion at m/z 1290.3±0.1 dalton. All other peaks above 800 m/z mass units (not counting isotope peaks) in the spectrum were lower than 20% of the molecular ion, upon analysis with a Kratos MS-50 double focusing mass spectrometer under the following experimental conditions: Xe fast atom bombardment at 6 Kv; glycerol matrix; positive ionization mode

I) The aminoacid analysis of the hydrochloric acid hydrolysate shows the presence of the following natural aminoacids: glycine, (L)proline and (L)serine, under the following experimental conditions:

the sample is hydrolyzed at 105° C. for 20 hours in the presence of 6N HCl containing 1% phenol and then derivatized in two steps as follows:

a) formation of the n-propyl esters of the carboxylic acid functions with 2M HCl in anhydrous pronapol (90° C., 1 h), and followed by drying under nitrogen;

b) conversion of the free amino groups to amides with pentafluoropropionic anhydride/anhydrous dichloromethane, 1/9 (v/v) at room temperature for 1 h followed by drying under nitrogen;

the derivatized residue so obtained is dissolved in dichloromethane and analyzed by GC-MS using a HP5985B system under the following conditions: column: chiral n-propionyl-L-valine t-butylamide polysiloxane coated fused silica capillary column (25 m×0.2 mm i.d.; C.G.C. ANALYTIC); temperature program 80° C. for 4 min, then 4° C./min

L) The ionization studies do not show any ionizable functions by titration with 0.1N HCl and 0.1N NaOH in Methylcellosolve/water; a weak basic function is revealed by titration with 0.1N HClO₄ in a non-aqueous medium (acetic acid);

M) The specific rotation value α!_(D) ²⁰ is +140.8; measured in absolute ethanol, at a concentration of about 5 gr/l. 

We claim:
 1. A method for the treatment of bacterial infections comprising administering to a patient with a bacterial infection an antibacterially effective amount of a single antibiotic substance, in which said single antibiotic substance is selected from the group consisting of antibiotic GE 2270 factor B₁, antibiotic GE 2270 factor B₂, antibiotic GE 2270 factor C₁, antibiotic GE 2270 factor C₂, antibiotic GE 2270 factor D₁, antibiotic GE 2270 factor D₂, antibiotic GE 2270 factor E, and antibiotic GE 2270 factor T; in which each of said single antibiotic substances has the following characteristics:A) Ultraviolet absorption spectra exhibiting the following maxima:

    ______________________________________                                                           λ max (nm)                                            ______________________________________                                         ANTIBIOTIC GE 2270 FACTORS B.sub.1, B.sub.2, C.sub.1, C.sub.2, D.sub.1,        D.sub.2 AND E:                                                                 0.1M HCl            245-250 (shoulder)                                                             300-315                                                    0.1M KOH            245-250 (shoulder)                                                             300-315                                                    Phosphate buffer pH 7.38                                                                           245-250 (shoulder)                                                             300-315                                                    Methanol            245-250 (shoulder)                                                             300-315                                                    ANTIBIOTIC GE 2270 FACTORS T:                                                  0.1 M HCl           250                                                                            280 (shoulder)                                                                 345 (shoulder)                                             0.1M KOH            240 (shoulder)                                                                 290 (shoulder)                                                                 345 (shoulder)                                             Phosphate buffer pH 7.38                                                                           250 (shoulder)                                                                 310 (shoulder)                                                                 345 (shoulder)                                             Methanol            240 (shoulder)                                                                 275 (shoulder)                                                                 290 (shoulder)                                                                 311                                                                            335 (shoulder)                                             ______________________________________                                    

B) Infrared absorption spectra (antibiotic GE 2270 factors D₁, D₂, E and T) in nujol mull exhibiting the following absorption maxima ν(cm⁻¹):Factor D₁ : 3700-3100; 3020-2750 (nujol); 1645; 1570-1490; 1460 and 1375 (nujol); 1305; 1260-1100; 1100-870; 840; 800; 760; 720 (nujol); 700 Factor D₂ : 3700-3100; 3020-2750 (nujol); 1645; 1570-1490; 1460 and 1375 (nujol); 1305; 1260-1100; 1100-870; 840; 800; 760; 720 (nujol); 700 Factor E: 3700-3100; 3020-2750 (nujol); 1645; 1570-1490; 1460 and 1375 (nujol); 1305; 1260; 1250-1070; 1015; 985-900; 840; 800; 760; 740; 720 (nujol); 700 Factor T: 3700-3120; 3100; 3020-2750 (nujol); 1655; 1570-1490; 1460 and 1375 (nujol); 1410; 1300; 1240; 1200-1000; 980; 930; 890; 840; 805; 765; 745; 720 (nujol); 700 C) ¹ H-N spectrum (antibiotic GE 2270 factors D₁, D₂, E and T) exhibiting the followoing groups of signals in DMSO-d₆ (hexadeuterodimethyl-sulfoxide) using TMS as the internal standard (0.00 ppm) (s=singlet, br s=broad singlet, d=doublet, dd=doublet of doublets, t=triplet, m=multiplet, Py=pyridine, Tz=thiazole)Factor D₁ (recorded at 500 MHz): 888, d, H); 8.70, d (2NH's); 8.57, s, 8.50, s, 8.25, s, 8.21, s and 7.35 (s, (CH's of 5 thizoles); 8.40, m, (glycine NH); 8.28-8.21, m, (pyridine CH's); 7.32-7.20, m, (aromatic CH's and primary amide NH); 7.00, s, 6.64, s, 6.53, s (primary amide NH's); 5.95, d, (OH); 5.29-5.15, m, (αCH's); 5.04, m, (phenylserine βCH); 4.81, m and 4.56, m, (oxazoline CH₂); 4.30-3.80, m, (CH₂ of glycine and prolineamide CH's); 2.72, m, and 1.43, m, (CH₂ of asparagine); 2.60, s, (CH₃); 2.21-1.91, (m), (isopropyl CH and prolineamide CH's); 0.90 (d) and 0.86 (d), (valine CH₃) Factor D₂ (recorded at 500 MHz): 9.00, d, (NH); 8.69, br s (2NH's); 8.59, s, 8.53, s, 8.29, s and 7.35, s, (thiazole CH's); 8.38, m, (glycine NH); 8.40 and 8.26 (m), (Py.CH's); 7.37-7.18, m, (aromatic CH's, primary amide NH); 6.97, s, (primary amide NH); 6.03, d and t, (2 OH's); 5.28-5.16, m (αCH's); 5.03, m, (βCH); 4.97, m, CH₂ (OH)!; 4.79 and 4.55, m (CH₂ of oxazoline); 3.97-3.76, m, (CH₂ of glycine and CH's of prolineamide); 2.71, m and 1.28, m, (CH₂ of N-methylasparagine); 2.18-1.89, m, (isopropyl CH and prolineamide (CH₂ 's); 0.88, d and 0.84 d (valine CH₃ 's) Factor E (recorded at 500 MHz): 8.95, d, (NH); 8.73, d, (NH); 8.60, s, (Tz.CH ); 8.57, d, (NH ); 8.53, s, (Tz.CH); 8.42, m, (Py.CH's); 8.31, m, H); 8.28, m, (Py.CH); 8.24, s, (Tz.CH); 7.33, s, (Tz.CH); 7.31-7.20, m, (aromatic CH's, primary amide NH); 6.98, s, 6.91, s, 6.62, s, (primary amide NH's); 6.04, d, (OH); 5.95, t, (OH); 5.28-5.14, m (αCH's), 5.03, m, (βCH); 4.99, m, CH₂ (OH)!; 4.81, dd and 4.57, dd, (oxazoline CH₂); 4.26 (m) and 3.79 (m), (glycine CH₂); 4.25, m, 3.98, m, 3.82, m, (prolineamide CH's); 2.77, m, and 1.25, m, (CH₂ of asparagine); 2.60, s, (CH₃); 2.20, m, and 1.89, m, (valine βCH and CH₂ of prolineamide); 0.90, d, 0.84, d, (valine CH₃ 's) Factor T (recorded at 250 MHz): 8.95, d, (H); 8.70, d, (2NH's); 8.66, s, 8.65, s, 8.60, s, 8.30, s and 7.38, s, (CH's or 4 thiazoles and 1 oxazole); 7.35-7.24, m, (aromatic CH's and primary amide NH); 6.68 (primary amide NH); 5.96, d, (OH); 5.34-5.18, m (αCH's); 5.05, m, (βCH); 5.03, s, CH₂ (OCH₃)!; 4.32, m and 3.82, m, (CH₂ of glycine); 4.48, m, 4.04, m and 3.63, m, (CH's of prolineamide); 3.40, s, (OCH₃); 2.73, m, and 1.41, m, (CH₂ of N-methylasparagine); 2.60, s, (CH₃); 2.49, d, (CH₃ of N-methylasparagine); 2.27-1.88, m (isopropyl CH and CH₂ of prolineamide); 0.89, d, (CH₃ of valine) D) Retention times (R_(t)) (antiobiotic GE 2270 factors B₁, B₂, C₁, C₂, D₁, D₂, E and T) in the following reverse phase HPLC system:Column: Bakerbond® C8 (5 μm) 4.6×250 mm (Bakerbond is a trade name for reverse phase octylsilyl silica gel HPLC columns supplied by J. T. Baker Research Product, Phillisburg, N.J. 08865 USA) Flow rate: 1.8 ml/min Phase A: CH₃ CN:tetrahydrofuran:40 mM HCOONH₄ 40:40:20 Phase B: CH₃ CN:tetrahydrofuran:40 mM HCOONH₄ 10:10:80 Elution: linear gradient from 20% to 30% of Phase A in 20 min Detection: UV 254 nm are reported below:

    ______________________________________                                                         Retention time                                                 Antibiotic GE   relative to factor                                                                         R.sub.t                                            2270 factor     A (R.sub.t 16.6 min)                                                                       (min)                                              ______________________________________                                         E               0.52        8.62                                               D.sub.1         0.56        9.36                                               D.sub.2         0.59        9.72                                               C.sub.1         0.65        10.78                                              C.sub.2         0.79        13.10                                              B.sub.1         0.85        14.18                                              B.sub.2         0.97        16.08                                              T               1.19        19.68                                              ______________________________________                                    

E) The main FAB-MS peaks are indicated below:

    ______________________________________                                         Antibiotic GE  FAB-MS                                                          2270 factor    M + 1 ion                                                       ______________________________________                                         E              1262                                                            D.sub.1        1232                                                            D.sub.2        1276                                                            C.sub.1        1246                                                            C.sub.2        1276                                                            B.sub.1        1276                                                            B.sub.2        1260                                                            T              1288                                                            ______________________________________                                    

and in which said single antibiotic substance GE 2270 factors B₁, B₂, C₁, C₂, D₁, D₂, E and T is produced by:1) cultivating Planobispora rosea ATCC 53773, under aerobic conditions in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen, and inorganic salts, for a period of time sufficient to produce the crude antibiotic GE 2270 complex containing GE 2270 factors B₁, B₂, C₁, C₂, D₁, D₂, E and T; 2) recovering said crude antibiotic GE 2270 complex containing GE 2270 factors B₁, B₂, C₁, C₂, D₁, D₂, E and T from the medium; 3) isolating said single crude GE 2270 factors B₁, B₂, C₁, C₂, D₁, D₂, and E from said crude antibiotic GE 2270 complex containing GE 2270 factors B₁, B₂, C₁, C₂, D₁, D₂, E and T by high performance liquid chromatography employing silica gel and eluting first with methylene chloride and then sequentially with mixtures of methylene chloride and methanol in the following ratios: 98/2; 96/4, 94/6, 92/8, 90/10 and 88/12 (v/v) and pooling fractions according to their antibiotic content; 4) isolating said single crude GE 2270 factor T from said crude antibiotic GE 2270 complex containing GE 2270 factors B₁, B₂, C₁, C₂, D₁, D₂, E and T by high performance liquid chromatography employing silica gel and eluting with a 93:7 mixture of CH₂ Cl₂ :methanol, and pooling the fractions containing it; 5) separating and isolating said single antibiotic substance GE 2270 factors B₁, B₂, C₁, C₂, D₁, D₂, and E from the individual pooled fractions of single crude GE 2270 factors B₁, B₂, C₁, C₂, D₁, D₂, and E by high performance liquid chromatography employing an octadecyl functionalized silica gel and eluting with a 26:74 mixture of Phase A: CH₃ CN:tetrahydrofuran:40 mM HCOONH₄ (40:40:20) and Phase B: CH₃ CN:tetrahydrofuran:40 mM HCOONH₄, collecting according to the UV adsorption profile at 254 nm; 6) separating and isolating said single antibiotic substance GE 2270 factor T from the individual pooled fraction containing single crude GE 2270 factor T by high performance liquid chromatography employing an octadecyl functionalized silica gel and eluting with a 60:40 mixture of CH₃ CN:H₂ O and collecting according to UV adsorption profile at 254 nm. 